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Enzyme Kinetics

The apparent kinetic constants (KM, Vmax, Clint) are determined using microsomes, primary hepatocytes or recombinant enzymes.

Quantity of test item required: 5 mg
Turnaround time of draft report: 20 working days after study initiation

Range Finding

  • single determinations
  • three concentrations of test system (e.g. 0.5, 1,1.5 mg microsomal protein/ml)
  • three concentrations of test item (e.g. 1, 10, 100 µM)
  • three time points (e.g. 15, 30, 60 min)
  • positive control: enzyme marker reaction (e.g. formation of 7-hydroxycoumarin)
  • negative control: zero time point and no NADPH

Study Design:

  • triplicates
  • one concentration of test item
  • seven to ten concentrations of test item
  • one time point
  • positive control: enzyme marker reaction
  • negative control: zero time point


  • HPLC-DAD/FLD or LC-MS/MS or Radiodetection:
    detection of loss of parent compound and/or formation of selected metabolites, quantitative


If no comparable data are available, a range-finding pretest is pre-requisite for the determination of the enzyme kinetics in order to ensure sufficient metabolism of the test item as well as linear response with time and test system concentration.
Further, a reference metabolite should be available for calibration and calculation of the Vmax and Clint. Alternatively, radiolabelled test item can be used. Indirect calculation by the loss of parent compound is usually limited by insufficient metabolism rates at high test item concentrations.

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GenPharmTox BioTech AG • phone: +49 (0) 89 89 55 59-0 • fax: +49 (0) 89 89 55 59-18 • eMail: info@genpharmtox.de

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